To identify novel therapeutic targets to reverse inflammatory cytokine effects on behavior, this study will explore mechanisms by which interferon (IFN)-alpha affects CNS glutamate using post-mortem brain tissue collected from a back translational model of cytokine-induced depressive-like behavior. Inflammatory cytokines and their signaling pathways are reliably elevated in a significant proportion of depressed patients, and administration of cytokines is associated with development of depressive symptoms in laboratory animals and humans. For instance, peripheral administration of IFN-alpha for treatment of hepatitis C and malignant melanoma is well known to induce clinical depression in up to 50% of patients, and has been shown to cause anhedonic and depressive-like behavior in laboratory animals. Despite the mounting evidence that inflammatory cytokines affect behavior, the CNS mechanisms by which cytokines cause depressive symptoms are only beginning to be understood. One pathway that is receiving increasing attention is CNS glutamate. Recent results from our group using magnetic resonance spectroscopy indicate that administration of IFN-alpha to patients with hepatitis C increased glutamate in the basal ganglia and anterior cingulate cortex (ACC), which correlated with depressive symptoms. In addition, our preliminary results from RNA sequencing in postmortem tissue demonstrate that peripheral administration of IFN-alpha not only decreased in vivo expression of the glutamate transporter (excitatory amino acid transporter 2), but also increased in vivo gene expression relevant to glutamate neurotransmission, including N-methyl-D-aspartate (NMDA) receptor subunits and metabotropic glutamate receptors that are involved in glutamate excitotoxicity. IFN-alpha may also increase glutamate neurotransmission through activation of the kynurenine pathway and production of quinolinic acid (QUIN), an NMDA receptor agonist and neurotoxin, by activated microglia and CNS macrophages. Of note, our preliminary data indicate that IFN-alpha increased perivascular macrophages and increased microglia activation in specific basal ganglia nuclei. Based on these findings from animals and humans, this proposal will test the hypothesis that chronic IFN-alpha increases glutamate and affects glutamate neurotransmission in the basal ganglia and ACC through decreasing glutamate transporters, increasing excitotoxic glutamate receptor subtypes and their signaling pathways, and increasing the production of QUIN by activated microglia and perivascular macrophages. This study will be the first to elucidate cytokine-induced changes in glutamate neurotransmission in vivo and link these changes to QUIN induction by activated macrophages and microglia in an established, back translational model. This study will also reveal new treatment strategies for future clinical trials to reverse or prevent depressive symptoms in patients exposed to elevated levels of inflammatory cytokines.